The transforming growth factor beta (TGF-β) superfamily of cytokines regulates a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development, production of extracellular matrix, and the immune response. Each subgroup of this superfamily initiates a unique intracellular signaling cascade activated by ligand-induced formation and activation of specific serine/threonine kinase receptor complexes. In mammals, the TGF-β subfamily comprises three transforming growth factor beta isoforms, TGF-β1, TGF-β2, and TGF-β3. Signal transduction occurs through a requisite interaction between TGF-β type I and II receptors. Once transforming growth factor-beta 3 (also known as TGF-β3, TGF-beta-3, Tgfb3, and TGFB3) complexes with the type I and II TGF-β receptors, a phosphorylation cascade is initiated and sent to cytoplasmic effector molecules, the Smad proteins, for propagation of the kinase signal to nuclear transcription factors (Piek et al., Faseb J., 1999, 13, 2105-2124).
Transforming growth factor-beta 3 was cloned from the human A673 rhabdomyosarcoma cell line (ten Dijke et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 4715-4719) and mapped to the 14q24 locus, a region associated with familial Alzheimer's disease (FAD) (Ardley et al., Cytogenet. Cell Genet., 1998, 82, 107-109.).
Transforming growth factor-beta 3 is believed to have a role in healing of wounds and bone fractures, and is not expressed in healthy skin. It can function as a morphogen when injected subperiosteally, inducing cartilage and bone formation (Rosier et al., Clin. Orthop., 1998, S294-300). The expression pattern of transforming growth factor-beta 3 differs from that of the other TGF-β isoforms, which are believed to play distinct and nonredundant functions in wound healing. Upon wounding, expression of transforming growth factor-beta 3 is upregulated. However, in the absence of TGF-β1, transforming growth factor-beta 3 expression is delayed, leading to delayed wound healing (Crowe et al., J. Invest. Dermatol., 2000, 115, 3-11).
Transforming growth factor-beta 3 also appears to play a role in orofacial and lung development, as allelic variants have been linked to the occurrence of cleft lip and palate in mice and humans (Kaartinen et al., Nat. Genet., 1995, 11, 415-421; Romitti et al., Teratology, 1999, 59, 39-50). A transforming growth factor-beta 3 null mouse has been generated and homozygous Tgfb3 (−/−) mice have an incompletely penetrant cleft palate and a delay in pulmonary development, implicating transforming growth factor-beta 3 in defective palatogenesis and lung morphogenesis and suggesting involvement of this cytokine in epithelial-mesenchymal interaction (Kaartinen et al., Nat. Genet., 1995, 11, 415-421).
A failure to downregulate the expression of transforming growth factor-beta 3 at 9-weeks gestation is believed to predispose human pregnancies to preeclampsia, a condition which results from insufficient invasion of the maternal decidua by placental extravillous trophoblasts (Caniggia et al., J. Clin. Invest., 1999, 103, 1641-1650).
Mutant function or overactivity of TGF-β signaling components have been implicated in cancers of the colon, esophagus, pancreas, lung, and breast, as well as in hyperproliferative disorders of the kidney, atherosclerosis, and rheumatoid arthritis (Markowitz, J. Clin. Invest., 1997, 100, 2143-2145; Pasche, J. Cell Physiol., 2001, 186, 153-168; Piek et al., Faseb J., 1999, 13, 2105-2124; Schuppan et al., Acta Gastroenterol. Belg., 2000, 63, 366-370). Specifically, upregulation of transforming growth factor-beta 3 expression has been demonstrated in hepatocellular carcinoma cells and perineoplastic stroma of the liver, suggesting a role in tumor progression (Abou-Shady et al., Am. J. Surg., 1999, 177, 209-215). Furthermore, increased expression of transforming growth factor-beta 3 in breast cancer patients is a prognostic indicator inversely correlated with survival (Ghellal et al., Anticancer Res., 2000, 20, 4413-4418).
Chronic diseases of the liver, pancreas, intestine, kidneys, skin, and lungs often lead to organ fibrosis and scarring, and progressive loss of organ function, despite the use of anti-viral or anti-inflammatory agents. TGF-βs are considered to be the most potent fibrogenic cytokines, and thus, the modulation of transforming growth factor-beta 3 activity and/or expression is an ideal target for therapeutic intervention in the prevention and treatment of fibroproliferative diseases (Schuppan et al., Acta Gastroenterol. Belg., 2000, 63, 366-370).
Investigative strategies aimed at modulating expression of transforming growth factor-beta 3 and studying its function have involved the use of polyclonal antibodies and antisense oligonucleotides.
A phosphorothioate antisense oligodeoxynucleotide, 17 nucleotides in length, spanning the initiation codon of chicken transforming growth factor-beta 3 mRNA was used to show that transforming growth factor-beta 3 functions in an autocrine fashion in the atrioventricular canal endocardium during chick heart development (Nakajima et al., Developmental Biology, 1998, 194, 99-113).
One antisense oligonucleotide, 16 nucleotides in length, hybridizing to four codons 5′ to the initiation site and including the initiation codon of the mouse transforming growth factor-beta 3 mRNA sequence, was used to show that transforming growth factor-beta 3 regulates embryonic Meckel's cartilage and tooth development (Chai et al., Dev. Biol., 1994, 162, 85-103), and that transforming growth factor-beta 3 is induced by glucocorticoids in fetal rat lung fibroblasts (Yee et al., Am. J. Physiol., 1996, 270, L992-1001) but does not inhibit rat lung branching in vitro (Liu et al., Dev. Dyn., 2000, 217, 343-360).
Finally, this same antisense oligonucleotide was also used to show that inhibition of transforming growth factor-beta 3 restores the invasive capability of human trophoblasts into the maternal decidua in preeclamptic pregnancies (Caniggia et al., J. Clin. Invest., 1999, 103, 1641-1650).
Disclosed and claimed in PCT Publication WO 94/25588 are phosphorothioate antisense oligonucleotides wherein said oligonucleotides hybridize with an area of a gene coding for transforming growth factor-beta 3 (Schlingensiepen et al., 1994).
Disclosed and claimed in PCT Publication WO 99/63975 are medicaments comprising a combination of at least one inhibitor of TGF-β family members and their receptors, wherein the inhibitor is an antisense nucleotide and/or ribozyme, as well as methods of using said medicaments in the treatment of neoplasms or infectious diseases (Schlingensiepen et al., 1999).
Disclosed and claimed in PCT Publication WO 98/40747 are methods for regulating or increasing trophoblast invasion in a subject comprising administration of therapeutically effective amounts of an inhibitor of transforming growth factor-beta 3 wherein the inhibitor is antisense to transforming growth factor-beta 3 (Caniggia et al., 1998).
Currently, there are no known therapeutic agents that effectively inhibit the synthesis of transforming growth factor-beta 3. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting transforming growth factor-beta 3 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and therefore may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of transforming growth factor-beta 3 expression.
The present invention provides compositions and methods for modulating transforming growth factor-beta 3 expression, including modulation of the truncated mutants and the alternatively spliced isoforms of transforming growth factor-beta 3.